Coupled Sensing of Hunger and Thirst Signals Balances Sugar and Water Consumption.

Hunger and thirst are ancient homeostatic drives for food and water consumption. Although molecular and neural mechanisms underlying these drives are currently being uncovered, less is known about how hunger and thirst interact. Here, we use molecular genetic, behavioral, and anatomical studies in Drosophila to identify four neurons that modulate food and water consumption. Activation of these neurons promotes sugar consumption and restricts water consumption, whereas inactivation promotes water consumption and restricts sugar consumption. By calcium imaging studies, we show that these neurons are directly regulated by a hormone signal of nutrient levels and by osmolality. Finally, we identify a hormone receptor and an osmolality-sensitive ion channel that underlie this regulation. Thus, a small population of neurons senses internal signals of nutrient and water availability to balance sugar and water consumption. Our results suggest an elegant mechanism by which interoceptive neurons oppositely regulate homeostatic drives to eat and drink.

Contact chemoreceptors mediate male-male repulsion and male-female attraction during Drosophila courtship.

The elaborate courtship ritual of Drosophila males is dictated by neural circuitry established by the transcription factor Fruitless and triggered by sex-specific sensory cues. Deciphering the role of different stimuli in driving courtship behavior has been limited by the inability to selectively target appropriate sensory classes. Here, we identify two ion channel genes belonging to the degenerin/epithelial sodium channel/pickpocket (ppk) family, ppk23 and ppk29, which are expressed in fruitless-positive neurons on the legs and are essential for courtship. Gene loss-of-function, cell-inactivation, and cell-activation experiments demonstrate that these genes and neurons are necessary and sufficient to inhibit courtship toward males and promote courtship toward females. Moreover, these cells respond to cuticular hydrocarbons, with different cells selectively responding to male or female pheromones. These studies identify a large population of pheromone-sensing neurons and demonstrate the essential role of contact chemosensation in the early courtship steps of mate selection and courtship initiation.

Pregnancy health in a multi-state U.S. population of systemically underserved patients and their children: PROMISE cohort design and baseline characteristics.

BACKGROUND: Gestational weight gain (GWG) is a routinely monitored aspect of pregnancy health, yet critical gaps remain about optimal GWG in pregnant people from socially marginalized groups, or with pre-pregnancy body mass index (BMI) in the lower or upper extremes. The PROMISE study aims to determine overall and trimester-specific GWG associated with the lowest risk of adverse birth outcomes and detrimental infant and child growth in these underrepresented subgroups. This paper presents methods used to construct the PROMISE cohort using electronic health record data from a network of community-based healthcare organizations and characterize the cohort with respect to baseline characteristics, longitudinal data availability, and GWG. METHODS: We developed an algorithm to identify and date pregnancies based on outpatient clinical data for patients 15 years or older. The cohort included pregnancies delivered in 2005-2020 with gestational age between 20 weeks, 0 days and 42 weeks, 6 days; and with known height and adequate weight measures needed to examine GWG patterns. We linked offspring data from birth records and clinical records. We defined study variables with attention to timing relative to pregnancy and clinical data collection processes. Descriptive analyses characterize the sociodemographic, baseline, and longitudinal data characteristics of the cohort, overall and within BMI categories. RESULTS: The cohort includes 77,599 pregnancies: 53% had incomes below the federal poverty level, 82% had public insurance, and the largest race and ethnicity groups were Hispanic (56%), non-Hispanic White (23%) and non-Hispanic Black (12%). Pre-pregnancy BMI groups included 2% underweight, 34% normal weight, 31% overweight, and 19%, 8%, and 5% Class I, II, and III obesity. Longitudinal data enable the calculation of trimester-specific GWG; e.g., a median of 2, 4, and 6 valid weight measures were available in the first, second, and third trimesters, respectively. Weekly rate of GWG was 0.00, 0.46, and 0.51 kg per week in the first, second, and third trimesters; differences in GWG between BMI groups were greatest in the second trimester. CONCLUSIONS: The PROMISE cohort enables characterization of GWG patterns and estimation of effects on child growth in underrepresented subgroups, ultimately improving the representativeness of GWG evidence and corresponding guidelines.

Taste adaptations associated with host specialization in the specialist Drosophila sechellia.

Chemosensory-driven host plant specialization is a major force mediating insect ecological adaptation and speciation. Drosophila sechellia, a species endemic to the Seychelles islands, feeds and oviposits on Morinda citrifolia almost exclusively. This fruit is harmless to D. sechellia but toxic to other Drosophilidae, including the closely related generalists D. simulans and D. melanogaster, because of its high content of fatty acids. While several olfactory adaptations mediating D. sechellia's preference for its host have been uncovered, the role of taste has been much less examined. We found that D. sechellia has reduced taste and feeding aversion to bitter compounds and host fatty acids that are aversive to D. melanogaster and D. simulans. The loss of aversion to canavanine, coumarin and fatty acids arose in the D. sechellia lineage, as its sister species D. simulans showed responses akin to those of D. melanogaster. Drosophila sechellia has increased taste and feeding responses towards M. citrifolia. These results are in line with D. sechellia's loss of genes that encode bitter gustatory receptors (GRs) in D. melanogaster. We found that two GR genes which are lost in D. sechellia, GR39a.a and GR28b.a, influence the reduction of aversive responses to some bitter compounds. Also, D. sechellia has increased appetite for a prominent host fatty acid compound that is toxic to its relatives. Our results support the hypothesis that changes in the taste system, specifically a reduction of sensitivity to bitter compounds that deter generalist ancestors, contribute to the specialization of D. sechellia for its host.

Role of Endothelial STAT3 in Cerebrovascular Function and Protection from Ischemic Brain Injury.

STAT3 plays a protective role against ischemic brain injury; however, it is not clear which brain cell type mediates this effect, and by which mechanism. We tested the hypothesis that endothelial STAT3 contributes to protection from cerebral ischemia, by preserving cerebrovascular endothelial function and blood-brain barrier (BBB) integrity. The objective of this study was to determine the role of STAT3 in cerebrovascular endothelial cell (EC) survival and function, and its role in tissue outcome after cerebral ischemia. We found that in primary mouse brain microvascular ECs, STAT3 was constitutively active, and its phosphorylation was reduced by oxygen-glucose deprivation (OGD), recovering after re-oxygenation. STAT3 inhibition, using two mechanistically different pharmacological inhibitors, increased EC injury after OGD. The sub-lethal inhibition of STAT3 caused endothelial dysfunction, demonstrated by reduced nitric oxide release in response to acetylcholine and reduced barrier function of the endothelial monolayer. Finally, mice with reduced endothelial STAT3 (Tie2-Cre; STAT3flox/wt) sustained larger brain infarcts after middle cerebral artery occlusion (MCAO) compared to wild-type (WT) littermates. We conclude that STAT3 is vital to maintaining cerebrovascular integrity, playing a role in EC survival and function, and protection against cerebral ischemia. Endothelial STAT3 may serve as a potential target in preventing endothelial dysfunction after stroke.

Associations between perfluoroalkyl substances and thyroid hormones after high exposure through drinking water.

BACKGROUND: The reported associations for several per- and polyfluoroalkyl substance (PFAS) with thyroid hormones are inconsistent in epidemiological studies. The purpose of the current study was to investigate the possible association of thyroid hormones in relation to serum levels of perfluorohexane sulfonate, perfluorooctane sulfonate and perfluorooctanoic acid, in a Swedish general population, highly exposed through contaminated drinking water, and if the associations with PFAS remained in a comparison to a reference group based only on residency in areas with contrasting PFAS levels. METHOD: 3297 participants from Ronneby, a municipality with drinking water highly contaminated by PFAS (exposed group), and a reference group (N = 226) from a nearby municipality with non-contaminated drinking water supply were included. Regression analysis was used to investigate the associations between PFAS exposure, assessed as exposure groups (Ronneby and reference groups) and measured serum PFAS levels, and thyroid hormone levels, with adjustments for age, sex and BMI. RESULT: No cross-sectional associations were found between PFAS and thyroid hormones in adults and seniors except for a positive association between PFAS and fT4 in males over 50. Higher thyroid hormone levels were found in the preteen children from Ronneby compared to the reference group. In contrast, within Ronneby, there was weak evidence of associations between increased PFAS levels and decreased fT3 in preteen boys, and decreased TSH in teenage males. No such pattern was found in preteen and teenage girls. CONCLUSION: The present study found no consistent evidence to support association of PFAS with thyroid hormones.

15 years of GDR: New data and functionality in the Genome Database for Rosaceae.

The Genome Database for Rosaceae (GDR, https://www.rosaceae.org) is an integrated web-based community database resource providing access to publicly available genomics, genetics and breeding data and data-mining tools to facilitate basic, translational and applied research in Rosaceae. The volume of data in GDR has increased greatly over the last 5 years. The GDR now houses multiple versions of whole genome assembly and annotation data from 14 species, made available by recent advances in sequencing technology. Annotated and searchable reference transcriptomes, RefTrans, combining peer-reviewed published RNA-Seq as well as EST datasets, are newly available for major crop species. Significantly more quantitative trait loci, genetic maps and markers are available in MapViewer, a new visualization tool that better integrates with other pages in GDR. Pathways can be accessed through the new GDR Cyc Pathways databases, and synteny among the newest genome assemblies from eight species can be viewed through the new synteny browser, SynView. Collated single-nucleotide polymorphism diversity data and phenotypic data from publicly available breeding datasets are integrated with other relevant data. Also, the new Breeding Information Management System allows breeders to upload, manage and analyze their private breeding data within the secure GDR server with an option to release data publicly.

Ranolazine may exert its beneficial effects by increasing myocardial adenosine levels.

We hypothesized that ranolazine-induced adenosine release is responsible for its beneficial effects in ischemic heart disease. Sixteen open-chest anesthetized dogs with noncritical coronary stenosis were studied at rest, during dobutamine stress, and during dobutamine stress with ranolazine. Six additional dogs without stenosis were studied only at rest. Regional myocardial function and perfusion were assessed. Coronary venous blood was drawn. Murine endothelial cells and cardiomyocytes were incubated with ranolazine and adenosine metabolic enzyme inhibitors, and adenosine levels were measured. Cardiomyocytes were also exposed to dobutamine and dobutamine with ranolazine. Modeling was employed to determine whether ranolazine can bind to an enzyme that alters adenosine stores. Ranolazine was associated with increased adenosine levels in the absence (21.7 ± 3.0 vs. 9.4 ± 2.1 ng/mL, P < 0.05) and presence of ischemia (43.1 ± 13.2 vs. 23.4 ± 5.3 ng/mL, P < 0.05). Left ventricular end-systolic wall stress decreased (49.85 ± 4.68 vs. 57.42 ± 3.73 dyn/cm2, P < 0.05) and endocardial-to-epicardial myocardial blood flow ratio tended to normalize (0.89 ± 0.08 vs. 0.76 ± 0.10, P = nonsignificant). Adenosine levels increased in cardiac endothelial cells and cardiomyocytes when incubated with ranolazine that was reversed when cytosolic-5'-nucleotidase (cN-II) was inhibited. Point mutation of cN-II aborted an increase in its specific activity by ranolazine. Similarly, adenosine levels did not increase when cardiomyocytes were incubated with dobutamine. Modeling demonstrated plausible binding of ranolazine to cN-II with a docking energy of -11.7 kcal/mol. We conclude that the anti-adrenergic and cardioprotective effects of ranolazine-induced increase in tissue adenosine levels, likely mediated by increasing cN-II activity, may contribute to its beneficial effects in ischemic heart disease.NEW & NOTEWORTHY Ranolazine is a drug used for treatment of angina pectoris in patients with ischemic heart disease. We discovered a novel mechanism by which this drug may exhibit its beneficial effects. It increases coronary venous levels of adenosine both at rest and during dobutamine-induced myocardial ischemia. Ranolazine also increases adenosine levels in endothelial cells and cardiomyocytes in vitro, by principally increasing activity of the enzyme cytosolic-5'-nucleotidase. Adenosine has well-known myocardial protective and anti-adrenergic properties that may explain, in part, ranolazine's beneficial effect in ischemic heart disease.

Inflammatory bowel disease and biomarkers of gut inflammation and permeability in a community with high exposure to perfluoroalkyl substances through drinking water.

Perfluoroalkyl substances (PFAS) can act as surfactants and have been suggested to be capable of affecting gut mucosa integrity, a possible factor in the pathogenesis of inflammatory bowel disease (IBD). So far, only PFOA has been shown to have a positive association with ulcerative colitis. The present study aimed to investigate the association of PFAS and clinically diagnosed IBD in the Ronneby cohort, a population with high PFAS exposure (especially high PFOS and PFHxS) from Aqueous Film-Forming Foam through drinking water, using registry data. Additionally, to explore associations of PFAS with fecal zonulin and calprotectin, subclinical biomarkers of gut inflammation and permeability, in a sub-set of participants from Ronneby and Karlshamn (a nearby control municipality). The registry study included all people that ever resided in Ronneby municipality at least one year between 1980 and 2013. Yearly exposure to contaminated drinking water was assessed based on residential addresses and waterworks supply data, and the population classified by early, mid and late periods in ascending level of contamination. Diagnosed IBD cases were retrieved from the Swedish National Patient register and cause-of-death register. The Cox proportional hazards model was used to derive the hazard ratios (HRs) for diagnosed IBD. The biomarker study included 189 individuals who provided fecal samples. Serum PFAS were measured using LC-MS/MS. Fecal zonulin and calprotectin were measured using ELISA. Linear regression was used to assess the associations between measured PFAS and biomarker levels. In the registry study, no raised HRs for diagnosed IBD were found for cohort subjects with mid (1995-2004) or late period (2005-2013) exposure compared to never exposure. Early period exposure only (1985-1994) showed raised HRs for Crohn's disease (HR = 1.58, p = 0.048) and other non-specified IBD (HR = 1.38, p = 0.037). In the biomarker study, Karlshamn showed higher fecal calprotectin levels (median = 99.6 mg/kg in Karlshamn vs. 66.8 mg/kg in Ronneby, p = 0.04). A trend of decreased calprotectin with increased serum PFAS indicated higher PFAS was associated with lower degree of gut inflammation (p = 0.002). No association between serum PFAS and fecal zonulin was found. In conclusion, the present study found no consistent evidence to support PFAS exposure as a risk factor for IBD.

Associations between perfluoroalkyl substances and serum lipids in a Swedish adult population with contaminated drinking water.

BACKGROUND: Exposures to perfluoroalkyl substances (PFAS) have shown positive associations with serum lipids in previous studies. While many studies on lipids investigated associations with perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), there are only a few studies regarding other PFAS, such as perfluorohexane sulfonic acid (PFHxS). The purpose of the current study is to investigate if associations with serum lipids were present, not only for serum PFOS and PFOA, but also for PFHxS, and if the associations with PFAS remained also in a comparison based only on residency in areas with contrasting exposure to PFAS. METHODS: 1945 adults aged 20-60 were included from Ronneby, Sweden, a municipality where one out of two waterworks had been heavily contaminated from aqueous fire-fighting foams, and from a nearby control area. The exposure was categorized based on either been living in areas with contrasting PFAS exposure or based on the actual serum PFAS measurements. Regression analyses of serum lipids were fitted against serum PFAS levels, percentile groups, smooth splines and between exposed and reference areas, adjusting for age, sex and BMI. RESULTS: Drinking water contamination caused high serum levels of PFOS (median 157 ng/ml) and PFHxS (median 136 ng/ml) and PFOA (median 8.6 ng/ml). These serum PFAS levels in the exposed groups were 5 to 100-fold higher than in the controls. In this population with mixed PFAS exposure, predominantly PFOS and PFHxS, PFAS exposure were positively associated with serum lipids. This was observed both when quantifying exposure as contrast between exposed and controls, and in terms of serum PFAS. Due to high correlations between each PFAS, we cannot separate them. CONCLUSIONS: In conclusion, the present study provides further evidence of a causal association between PFAS and serum lipids, especially for PFHxS.

Food-derived volatiles enhance consumption in Drosophila melanogaster.

Insects use multiple sensory modalities when searching for and accepting a food source, in particular odor and taste cues. Food-derived odorants are generally involved in mediating long- and short-range attraction. Taste cues, in contrast, act directly by contact with the food source, promoting the ingestion of nutritious food and the avoidance of toxic substances. It is possible, however, that insects integrate information from these sensory modalities during the process of feeding itself. Here, using a simple feeding assay, we investigated whether odors modulate food consumption in the fruit fly Drosophila melanogaster We found that the presence of both single food-derived odorants and complex odor mixtures enhanced consumption of an appetitive food. Feeding enhancement depended on the concentration and the chemical identity of the odorant. Volatile cues alone were sufficient to mediate this effect, as feeding was also increased when animals were prevented from contacting the odor source. Both males and females, including virgin females, increased ingestion in the presence of food-derived volatiles. Moreover, the presence of food-derived odorants significantly increased the consumption of food mixtures containing aversive bitter compounds, suggesting that flies integrate diverse olfactory and gustatory cues to guide feeding decisions, including situations in which animals are confronted with stimuli of opposite valence. Overall, these results show that food-derived olfactory cues directly modulate feeding in D. melanogaster, enhancing ingestion.

High exposure to perfluorinated compounds in drinking water and thyroid disease. A cohort study from Ronneby, Sweden.

Per- and polyfluoroalkyl substances (PFAS) are extremely persistent manmade substances. Apart from exposure through food and indoor air and dust, humans can be exposed through drinking water if the surface or groundwater is contaminated. In 2013 very high levels of PFOS and PFHxS were found in the drinking water from one of the two waterworks supplying the municipality of Ronneby, Sweden. A cohort was formed, including all individuals who had lived at least one year in Ronneby during the period 1980-2013 (ñ63,000). Each year, addresses that got their drinking water from the contaminated water works were identified. Through the Swedish personal identity number, each individual was linked to registers providing diagnoses and prescriptions for hyper- and hypothyroidism. In total, 16,150 individuals had ever been exposed. The hazard ratios did not indicate any excess risk of hyperthyroidism among those with contaminated water. For hypothyroidism, the risk of being prescribed medication was significantly increased among women with exposure during the mid part of the study period (but not men). However, the association with period of exposure was non-monotonic, so the significance is considered to be a chance finding. Our research was limited by the relatively simple exposure assessment.

Gustatory Processing in Drosophila melanogaster.

The ability to identify nutrient-rich food and avoid toxic substances is essential for an animal's survival. Although olfaction and vision contribute to food detection, the gustatory system acts as a final checkpoint control for food acceptance or rejection. The vinegar fly Drosophila melanogaster tastes many of the same stimuli as mammals and provides an excellent model system for comparative studies of taste detection. The relative simplicity of the fly brain and behaviors, along with the molecular genetic and functional approaches available in this system, allow the examination of gustatory neural circuits from sensory input to motor output. This review discusses the molecules and cells that detect taste compounds in the periphery and the circuits that process taste information in the brain. These studies are providing insight into how the detection of taste compounds regulates feeding decisions.

Half-lives of PFOS, PFHxS and PFOA after end of exposure to contaminated drinking water.

BACKGROUND: Municipal drinking water contaminated with perfluorinated alkyl acids had been distributed to one-third of households in Ronneby, Sweden. The source was firefighting foam used in a nearby airfield since the mid-1980s. Clean water was provided from 16 December 2013. OBJECTIVE: To determine the rates of decline in serum perfluorohexane sulfonate (PFHxS), perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA), and their corresponding half-lives. METHODS: Up to seven blood samples were collected between June 2014 and September 2016 from 106 participants (age 4-84 years, 53% female). RESULTS: Median initial serum concentrations were PFHxS, 277 ng/mL (range 12-1660); PFOS, 345 ng/mL (range 24-1500); and PFOA, 18 ng/mL (range 2.4-92). The covariate-adjusted average rates of decrease in serum were PFHxS, 13% per year (95% CI 12% to 15%); PFOS, 20% per year (95% CI 19% to 22%); and PFOA, 26% per year (95% CI 24% to 28%). The observed data are consistent with a first-order elimination model. The mean estimated half-life was 5.3 years (95% CI 4.6 to 6.0) for PFHxS, 3.4 years (95% CI 3.1 to 3.7) for PFOS and 2.7 years (95% CI 2.5 to 2.9) for PFOA. The interindividual variation of half-life was around threefold when comparing the 5th and 95th percentiles. There was a marked sex difference with more rapid elimination in women for PFHxS and PFOS, but only marginally for PFOA. CONCLUSIONS: The estimated half-life for PFHxS was considerably longer than for PFOS and PFOA. For PFHxS and PFOS, the average half-life is shorter than the previously published estimates. For PFOA the half-life is in line with the range of published estimates.

Neocentromeres: a place for everything and everything in its place.

Centromeres are essential for chromosome inheritance and genome stability. Centromeric proteins, including the centromeric histone centromere protein A (CENP-A), define the site of centromeric chromatin and kinetochore assembly. In many organisms, centromeres are located in or near regions of repetitive DNA. However, some atypical centromeres spontaneously form on unique sequences. These neocentromeres, or new centromeres, were first identified in humans, but have since been described in other organisms. Neocentromeres are functionally and structurally similar to endogenous centromeres, but lack the added complication of underlying repetitive sequences. Here, we discuss recent studies in chicken and fungal systems where genomic engineering can promote neocentromere formation. These studies reveal key genomic and epigenetic factors that support de novo centromere formation in eukaryotes.

Odontogenic Fibromyxoma in a Cat: First Confirmed Case in This Species.

An inflammatory gingival mass surrounding resorbing teeth was diagnosed via biopsy in a 9-year-old domestic shorthair cat. A dorsal rim excision was performed to remove the entire mass with associated teeth and bone. Histopathological diagnosis of the en bloc tissue revealed an odontogenic fibromyxoma. Extensive literature review revealed few case reports of companion animals with this neoplasm, and none in a feline patient. This report documents the clinical presentation, diagnostic differentials, surgical therapy, and long-term follow-up of an odontogenic fibromyxoma in a cat.

Gustatory learning and processing in the Drosophila mushroom bodies.

The Drosophila mushroom bodies are critical association areas whose role in olfactory associative learning has been well characterized. Recent behavioral studies using a taste association paradigm revealed that gustatory conditioning also requires the mushroom bodies (Masek and Scott, 2010; Keene and Masek, 2012). Here, we examine the representations of tastes and the neural sites for taste associations in the mushroom bodies. Using molecular genetic approaches to target different neuronal populations, we find that the gamma lobes of the mushroom bodies and a subset of dopaminergic input neurons are required for taste associative learning. Monitoring responses to taste compounds in the mushroom body calyx with calcium imaging reveals sparse, taste-specific and organ-specific activation in the Kenyon cell dendrites of the main calyx and the dorsal accessory calyx. Our work provides insight into gustatory representations in the mushroom bodies, revealing the essential role of gustatory inputs not only as rewards and punishments but also as adaptive cues.

The molecular basis for water taste in Drosophila.

The detection of water and the regulation of water intake are essential for animals to maintain proper osmotic homeostasis. Drosophila and other insects have gustatory sensory neurons that mediate the recognition of external water sources, but little is known about the underlying molecular mechanism for water taste detection. Here we identify a member of the degenerin/epithelial sodium channel family, PPK28, as an osmosensitive ion channel that mediates the cellular and behavioural response to water. We use molecular, cellular, calcium imaging and electrophysiological approaches to show that ppk28 is expressed in water-sensing neurons, and that loss of ppk28 abolishes water sensitivity. Moreover, ectopic expression of ppk28 confers water sensitivity to bitter-sensing gustatory neurons in the fly and sensitivity to hypo-osmotic solutions when expressed in heterologous cells. These studies link an osmosensitive ion channel to water taste detection and drinking behaviour, providing the framework for examining the molecular basis for water detection in other animals.

Motor neurons controlling fluid ingestion in Drosophila.

Rhythmic motor behaviors such as feeding are driven by neural networks that can be modulated by external stimuli and internal states. In Drosophila, ingestion is accomplished by a pump that draws fluid into the esophagus. Here we examine how pumping is regulated and characterize motor neurons innervating the pump. Frequency of pumping is not affected by sucrose concentration or hunger but is altered by fluid viscosity. Inactivating motor neurons disrupts pumping and ingestion, whereas activating them elicits arrhythmic pumping. These motor neurons respond to taste stimuli and show prolonged activity to palatable substances. This work describes an important component of the neural circuit for feeding in Drosophila and is a step toward understanding the rhythmic activity producing ingestion.

Science in Suzhou: establishment and function of neural circuits.

The Cold Spring Harbour Asia conference on 'Assembly, Plasticity, Dysfunction and Repair of Neural Circuits' took place in Suzhou, China, in October 2011. Developmental, cell, molecular and systems neuroscientists convened to discuss the establishment, function and plasticity of neural circuits in diverse organisms.